1. Technical Information
The present method and test mixture relates to the detection of Staphylococcus aureus in a biological, environmental, or food sample, and more particularly to those methods and test mixtures utilizing reacting factors which the target microbe(s) can convert into a clot.
2. Background Information
Staphylococcus aureus (S. aureus) can be a virulent pathogen of animals and humans Moreover, it can cause severe food poisoning by the production of a toxin. Diseases caused by S. aureus cover a very wide clinical spectrum, from simple skin infections to life threatening infections of the bones, heart, and organs. Of particular concern is the recognition that S. aureus infection is common after surgery. It is also associated with intravenous tubing and other implants.
The bacterium S. aureus may be transmitted between healthy individuals by skin to skin contact, or from a commonly shared item or a surface (e.g., tanning beds, gym equipment, food handling equipment, etc.) where the transfer may be made to a subsequent person who uses the shared item or touches the surface. Of great medical concern is the recognition that healthy people entering hospitals may “carry” S. aureus (e.g., on their skin, or in their noses, etc.) without any signs or symptoms that they do so. In the presence of favorable conditions (often found in but not limited to hospitals), the S. aureus can activate and cause serious infection. In addition, S. aureus can also be a source of food poisoning, often caused by a food handler contaminating the food product (e.g., meat, poultry, eggs, salads containing mayonnaise, bakery products, dairy products, etc.)
There are two categories of S. aureus based on an individual clone's susceptibility to the class of antibiotics that began with methicillin. These are methicillin susceptible S. aureus (MSSA), and methicillin resistant S. aureus (MRSA). Until only a few years ago, MRSA was most commonly found in hospitals. Now, many are also present in the noses, skin, etc. of people in the non-hospital community. Moreover, these MRSA are increasingly causing serious infections in the community. MRSA is particularly serious because very few antibiotics (e.g., vancomycin) have been shown to be uniformly effective against MRSA.
The Center for Disease Control and Prevention actively surveys for the development of methicillin resistant S. aureus. In 2000, the Society for Healthcare Epidemiology of America guidelines recommended contact isolation for patients with MRSA. In addition to the morbidity and mortality caused by MRSA, it has been estimated that each case of infection costs at least $23,000. Accordingly, many hospitals and nursing homes proactively sample patients for MRSA [Clany, M., Active Screening in High-Risk units is an effective and cost-avoidant method to reduce the rate of methicillin-resistant Staphylococcus aureus infection in the hospital, Infection Control and Hospital Epidemiology 27:1009-1017, 2006].
Meyer et al. (U.S. Pat. No. 4,035,238) describes the use of a broth for the detection of S. aureus that utilized mannitol as a source of carbon and DNA methyl green as an indicator. Neither of these chemicals are coagulase reactive substrates.
Rambach (U.S. Pat. No. 6,548,268) employs at least two chromogenic agents in an agar medium: 5-bromo-6-chloro-indoxyl-phosphate; and 5-bromo-4-chloro-3-indoxyl glucose in the presence of deferoxamine. An individual colony hydrolyzing these substrates will produce colors that will mix with each other and not be independent of one another.
A large number of classical culturing procedures are utilized to detect MSSA and MRSA from human, animal, food, etc. samples. They have in common a basic medium with chemical inhibitors such as 6-8% sodium chloride, potassium tellurite, and a variety of antibiotics. For example Stevens and Jones described the use of a trehalose-mannitol-phosphatase agar [Stevens, D L and Jones, C. Use of trehalose-mannitol-phosphatase agar to differentiate Staphylococcus epidermidis and Staphylococcus saprophyticus from other coagulase-negative staphylococci, J. of Clin. Microbiology 20:977-980, 1984]. The use of mannitol as a carbon source and salt as a selective agent into an agar known as mannitol-salt agar has been commonly used in clinical laboratories [Baird, R. M. and W. H. Lee, Media used in the detection and enumeration of Staphylococcus aureus, Int. J. Food Microbiology. 26:209-211, 1995]. Within the prior art of culturing, it is generally accepted procedure to perform coagulase tests utilizing samples of S. aureus that are isolated in a pure culture.
The procedure “S. aureus ID” [Bio Merieux, La Balme Les Grottes, France] uses an alpha-glucosidase substrate in agar to detect S. aureus. A single substrate is utilized. [Perry, J. D. et al., Evaluation of S. aureus ID, a new chromogenic agar medium for detection of Staphylococcus aureus, J. Clin. Microbiology 41:5695-5698, 2003]. A variant of this medium, which contains added antibiotics and sodium chloride, is designed to detect MRSA [Perry et al., Development and evaluation of a chromogenic agar medium for methicillin-resistant Staphylococcus aureus, J. of Clin. Micro. 42:4519-4523, 2004].
Selepak and Witebsky disclose a study evaluating the inoculum size and lot-to-lot variability of the tube coagulase test for S. aureus. Specimens were collected and isolates were generated from the bacterial colonies on agar plates. Tubes containing anticoagulated rabbit coagulase plasma were inoculated with a part of, or more than one, staphylococcal colony from the isolates. The tubes were incubated and examined for the presence of clot. According to Selepak and Witebsky, “with some isolates and some lots of coagulase plasma, even a single colony [from the isolate] may not provide enough inoculum for a positive coagulase test”. Furthermore, Selepak and Witebsky state that “[e]xpressed more quantitatively, at least 108 organisms per ml should be used whenever possible for each coagulase tube test. Our data further suggest that S. aureus does not grow in coagulase plasma; therefore, the incubation of coagulase plasma for 18 to 24 h does not compensate for the use of small inoculum.” Thus, Selepak and Witebsky indicate that it is impractical, if not impossible, to detect the presence or absence of S. aureus in first generation biological specimen samples using a direct coagulase test.
[Selepak, S. T et al, “Inoculum Size and Lot-to-Lot Variation as Significant Variables in the Tube Coagulase Test for Staphylococcus aureus”, Journal of Clin. Microbiology, November 1985, p. 835-837]
It would, therefore, be desirable to provide a test mixture and a method that can rapidly detect S. aureus directly from a sample, one that does not require a skilled technician to perform the method, one that can be performed without the need to develop isolates from the specimen (i.e., one that can be performed on a “first generational” sample), and one that does not require a large concentration of S. aureus organisms to be accurate.